Role In Anaerobic Respiration Research Articles

Identification and Characterization of the Novel Subunit CcoM in the cbb3₃Cytochrome c Oxidase from Pseudomonas stutzeri ZoBell.

ABSTRACTCytochrome c oxidases (CcOs), members of the heme-copper containing oxidase (HCO) superfamily, are the terminal enzymes of aerobic respiratory chains. The cbb3-type cytochrome c oxidases (cbb3-CcO) form the C-family and have only the central catalytic subunit in common with the A- and B-family HCOs. In Pseudomonas stutzeri, two cbb3 operons are organized in a tandem repeat. The atomic structure of the first cbb3 isoform (Cbb3-1) was determined at 3.2 Å resolution in 2010 (S. Buschmann, E. Warkentin, H. Xie, J. D. Langer, U. Ermler, and H. Michel, Science 329:327–330, 2010, http://dx.doi.org/10.1126/science.1187303). Unexpectedly, the electron density map of Cbb3-1 revealed the presence of an additional transmembrane helix (TMH) which could not be assigned to any known protein. We now identified this TMH as the previously uncharacterized protein PstZoBell_05036, using a customized matrix-assisted laser desorption ionization (MALDI)–tandem mass spectrometry setup. The amino acid sequence matches the electron density of the unassigned TMH. Consequently, the protein was renamed CcoM. In order to identify the function of this new subunit in the cbb3 complex, we generated and analyzed a CcoM knockout strain. The results of the biochemical and biophysical characterization indicate that CcoM may be involved in CcO complex assembly or stabilization. In addition, we found that CcoM plays a role in anaerobic respiration, as the ΔCcoM strain displayed altered growth rates under anaerobic denitrifying conditions.

Evidence for function overlapping of CymA and the cytochrome bc1 complex in the Shewanella oneidensis nitrate and nitrite respiration

Shewanella oneidensis is an important model organism for its versatility of anaerobic respiration. CymA, a cytoplasmic membrane-bound tetraheme c-type cytochrome, plays a central role in anaerobic respiration by transferring electrons from the quinone pool to a variety of terminal reductases. Although loss of CymA results in defect in respiration of many electron acceptors (EAs), a significant share of the capacity remains in general. In this study, we adopted a transposon random mutagenesis method in a cymA null mutant to identify substituent(s) of CymA with respect to nitrite and nitrate respiration. A total of 87 insertion mutants, whose ability to reduce nitrite was further impaired, were obtained. Among the interrupted genes, the petABC operon appeared to be the most likely candidate given the involvement of the cytochrome bc1 complex that it encodes in electron transport. Subsequent analyses not only confirmed that the complex and CymA were indeed functionally overlapping in nitrate/nitrite respiration but also revealed that both proteins were able to draw electrons from ubiquinone and menaquinone. Furthermore, we found that expression of the bc1 complex was affected by oxygen but not nitrate or nitrite and by global regulators ArcA and Crp in an indirect manner.

Influence of the molybdenum cofactor biosynthesis on anaerobic respiration, biofilm formation and motility in Burkholderia thailandensis

Burkholderia thailandensis is closely related to Burkholderia pseudomallei, a bacterial pathogen and the causative agent of melioidosis. B. pseudomallei can survive and persist within a hypoxic environment for up to one year and has been shown to grow anaerobically in the presence of nitrate. Currently, little is known about the role of anaerobic respiration in pathogenesis of melioidosis. Using B. thailandensis as a model, a library of 1344 transposon mutants was created to identify genes required for anaerobic nitrate respiration. One transposon mutant (CA01) was identified with an insertion in BTH_I1704 (moeA), a gene required for the molybdopterin biosynthetic pathway. This pathway is involved in the synthesis of a molybdopterin cofactor required for a variety of molybdoenzymes, including nitrate reductase. Disruption of molybdopterin biosynthesis prevented growth under anaerobic conditions, when using nitrate as the sole terminal electron acceptor. Defects in anaerobic respiration, nitrate reduction, motility and biofilm formation were observed for CA01. Mutant complementation with pDA-17:BTH_I1704 was able to restore anaerobic growth on nitrate, nitrate reductase activity and biofilm formation, but did not restore motility. This study highlights the potential importance of molybdoenzyme-dependent anaerobic respiration in the survival and virulence of B. thailandensis.

The role of anaerobic respiration in the immobilization of uranium through biomineralization of phosphate minerals

Although bioreduction of uranyl ions (U(VI)) and biomineralization of U(VI)–phosphate minerals are both able to immobilize uranium in contaminated sediments, the competition between these processes and the role of anaerobic respiration in the biomineralization of U(VI)–phosphate minerals has yet to be investigated. In this study, contaminated sediments incubated anaerobically in static microcosms at pH 5.5 and 7.0 were amended with the organophosphate glycerol-2-phosphate (G2P) as sole phosphorus and external carbon source and iron oxides, sulfate, or nitrate as terminal electron acceptors to determine the most favorable geochemical conditions to these two processes. While sulfate reduction was not observed even in the presence of G2P at both pHs, iron reduction was more significant at circumneutral pH irrespective of the addition of G2P. In turn, nitrate reduction was stimulated by G2P at both pH 5.5 and 7.0, suggesting nitrate-reducing bacteria provided the main source of inorganic phosphate in these sediments. U(VI) was rapidly removed from solution in all treatments but was not reduced as determined by X-ray absorption near edge structure (XANES) spectroscopy. Simultaneously, wet chemical extractions and extended X-ray absorption fine structure (EXAFS) spectroscopy of these sediments indicated the presence of U–P species in reactors amended with G2P at both pHs. The rapid removal of dissolved U(VI), the simultaneous production of inorganic phosphate, and the existence of U–P species in the solid phase indicate that uranium was precipitated as U(VI)–phosphate minerals in sediments amended with G2P. Thus, under reducing conditions and in the presence of G2P, bioreduction of U(VI) was outcompeted by the biomineralization of U(VI)–phosphate minerals and U(VI) sorption at both pHs.

The periplasmic nitrate reductase nap is required for anaerobic growth and involved in redox control of magnetite biomineralization in Magnetospirillum gryphiswaldense.

The magnetosomes of many magnetotactic bacteria consist of membrane-enveloped magnetite crystals, whose synthesis is favored by a low redox potential. However, the cellular redox processes governing the biomineralization of the mixed-valence iron oxide have remained unknown. Here, we show that in the alphaproteobacterium Magnetospirillum gryphiswaldense, magnetite biomineralization is linked to dissimilatory nitrate reduction. A complete denitrification pathway, including gene functions for nitrate (nap), nitrite (nir), nitric oxide (nor), and nitrous oxide reduction (nos), was identified. Transcriptional gusA fusions as reporters revealed that except for nap, the highest expression of the denitrification genes coincided with conditions permitting maximum magnetite synthesis. Whereas microaerobic denitrification overlapped with oxygen respiration, nitrate was the only electron acceptor supporting growth in the entire absence of oxygen, and only the deletion of nap genes, encoding a periplasmic nitrate reductase, and not deletion of nor or nos genes, abolished anaerobic growth and also delayed aerobic growth in both nitrate and ammonium media. While loss of nosZ or norCB had no or relatively weak effects on magnetosome synthesis, deletion of nap severely impaired magnetite biomineralization and resulted in fewer, smaller, and irregular crystals during denitrification and also microaerobic respiration, probably by disturbing the proper redox balance required for magnetite synthesis. In contrast to the case for the wild type, biomineralization in Δnap cells was independent of the oxidation state of carbon substrates. Altogether, our data demonstrate that in addition to its essential role in anaerobic respiration, the periplasmic nitrate reductase Nap has a further key function by participating in redox reactions required for magnetite biomineralization.

The role of chaperones in the initial steps of bacterial oxidoreductase maturation

Bacterial oxidoreductases play a key role in anaerobic respiration and represent a group of enzymes with complex maturation pathways. A group of specific chaperones, or redox enzyme maturation proteins (REMP), have an essential role in the maturation of bioenergetics enzymes. While their role has yet to be defined, REMPs have been shown to interact tightly with the amino terminal signal peptide of oxidoreductase subunits. DmsD, the REMP associated with the oxidoreductase dimethyl sulfoxide (DMSO) reductase, has been putatively shown to interact with a number of proteins. These include the general chaperones DnaK, and Trigger Factor (TF), and also include MoeB, a protein involved in the molybdenum cofactor biosynthesis. An in vitro characterization of these protein‐protein interactions will improve our understanding of the early steps of oxidoreductase maturation, as well as provide further insight into the role of REMPs. Studies to date were done using an in vitro modified far‐western technique, a robust technique that quantitatively assesses protein‐protein interactions between folded and pure proteins. An interaction was found between MoeB and DmsD, but this technique failed to show an interaction between DnaK or TF and DmsD. Alternatively, both DnaK and TF were shown to interact with the DMSO reductase signal peptide. Size exclusion chromatography is used to confirm protein‐protein interactions as well as to determine if these proteins can interact concurrently or if binding excludes one or more proteins.

Reduction of iron (III) and humic substances plays a major role in anaerobic respiration in an Arctic peat soil

Arctic peat soils contain vast reserves of organic C and are largely anaerobic. However, anaerobic respiration, particularly the role of Fe(III) and humic substances as electron acceptors, is not well understood in such ecosystems. We investigated these processes in a drained thaw lake basin on the Arctic coastal plain near Barrow, Alaska. We measured concentrations of soluble Fe and other potential electron acceptors, described the microbial community, and performed experiments in the laboratory and field to measure net rates of Fe(III) reduction and the relationship of this process to C cycling. In most areas within the basin, aerobic conditions existed only in the upper few centimeters of soil, though oxygen penetrated deeper in raised areas, such as rims of ice wedge polygons. Concentrations of nitrate and sulfate in soil pore water were low or negligible. Soil pore water contained surprisingly high concentrations of Fe(II) and Fe(III), in the range of hundreds of μM, suggesting the presence of organic chelators. The solid phase contained substantial amounts of iron minerals, with a progressively reduced oxidation state throughout the growing season. The most abundant 16S rRNA sequence in our gene survey was closely related to the Fe(III)‐reducing bacterium, Rhodoferax ferrireducens, and other sequences closely related to Fe‐transforming bacteria were found. Field and laboratory incubations with soluble Fe(III) and the quinonic compound, AQDS (a common humic analog), stimulated respiration and verified that Fe(III) reduction occurs in these soils. We conclude that reduction of Fe(III) and humic substances are major metabolic pathways in this ecosystem.

Characterization of Shewanella oneidensis MtrC: a cell-surface decaheme cytochrome involved in respiratory electron transport to extracellular electron acceptors

MtrC is a decaheme c-type cytochrome associated with the outer cell membrane of Fe(III)-respiring species of the Shewanella genus. It is proposed to play a role in anaerobic respiration by mediating electron transfer to extracellular mineral oxides that can serve as terminal electron acceptors. The present work presents the first spectropotentiometric and voltammetric characterization of MtrC, using protein purified from Shewanella oneidensis MR-1. Potentiometric titrations, monitored by UV-vis absorption and electron paramagnetic resonance (EPR) spectroscopy, reveal that the hemes within MtrC titrate over a broad potential range spanning between approximately +100 and approximately -500 mV (vs. the standard hydrogen electrode). Across this potential window the UV-vis absorption spectra are characteristic of low-spin c-type hemes and the EPR spectra reveal broad, complex features that suggest the presence of magnetically spin-coupled low-spin c-hemes. Non-catalytic protein film voltammetry of MtrC demonstrates reversible electrochemistry over a potential window similar to that disclosed spectroscopically. The voltammetry also allows definition of kinetic properties of MtrC in direct electron exchange with a solid electrode surface and during reduction of a model Fe(III) substrate. Taken together, the data provide quantitative information on the potential domain in which MtrC can operate.

Carbon-nanotube-modified electrodes for the direct bioelectrochemistry of pseudoazurin

The bioelectrochemistry of the blue copper protein, pseudoazurin, at glassy carbon and platinum electrodes that were modified with single-wall carbon nanotubes (SWNTs) was investigated by multiple scan rate cyclic voltammetry. The protein showed reversible electrochemical behavior at both bare glassy carbon electrodes (GCEs) and SWNT-modified GCEs (SWNT|GCEs); however, direct electrochemistry was not observed at any of the platinum electrodes. The effect of the carbon nanotubes at the GCE was to amplify the current response 1000-fold (nA at bare GCE to µA at SWNT|GCE), increase the apparent diffusion coefficient D app of the solution-borne protein by three orders of magnitude, from 1.35 × 10−11 at bare GCE to 7.06 × 10−8 cm2 s-1 at SWNT|GCE, and increase the heterogeneous electron transfer rate constant k s threefold, from 1.7 × 10−2 cm s−1 at bare GCE to 5.3 × 10−2 cm s−1 at SWNT|GCE. Pseudoazurin was also found to spontaneously adsorb onto the nanotube-modified GCE surface. Well-resolved voltammograms indicating quasi-reversible faradaic responses were obtained for the adsorbed protein in phosphate buffer, with I pc and I pa values now greater than corresponding values for solution-borne pseudoazurin at SWNT|GCEs and with significantly reduced ΔE p values. The largest electron transfer rate constant of 1.7 × 10−1 cm s−1 was achieved with adsorbed pseudoazurin at the SWNT|GCE surface in deaerated buffer solution consistent with its presumed role in anaerobic respiration of some bacteria.

The Role of Anaerobic Respiration in Germinating Muskmelon Seeds

The Role of Anaerobic Respiration in Germinating Muskmelon Seeds

The Role of Anaerobic Respiration in Germinating Muskmelon Seeds

The Role of Anaerobic Respiration in Germinating Muskmelon Seeds